The central hypothesis of this research is that gene encoded microbicidal peptides released by Paneth cells contribute to innate immunity in the small intestine. The apically oriented secretory granules of Paneth cells contain a-defensins, termed cryptdins, that are potent microbicidal agents in vitro and homologs of agents that mediate intracellular non-oxidative microbial cell killing by phagocytic cells. The objective of this application is to investigate the biological activity and biochemical mechanism of action of these peptides as mediators of bacterial cell death. Antimicrobial peptides mediate innate immunity both in phagocytes and on mucosal surfaces. In the small intestine, Paneth cells release secretory granules rich in bactericidal a-defensin peptides, known as cryptdins. Cryptdins with N-terminally truncated residues and attenuated microbicidal activity have been purified from rinses of adult mouse small intestine. In particular, cryptdin-4 deficient in the N-- terminal glycine ((des-Gly)-cryptdin-4) lacks bactericidal activity against Gram-positive bacteria compared to native cryptdin-4. This finding implicate the cryptdin-4 N-terminus as one determinant of microbicidal activity which has lead to the overall experimental goal of this proposal: To investigate the role of specific amino acid side chains in the primary sequence of cryptdin-4 on the bactericidal activity of this peptide as a means of defining its mechanism of action.